Protein and gene involved in myocyte differentiation

ABSTRACT

The invention relates to antibodies that bind to novel polypeptides expressed in immortalized cells, skeletal muscles and undifferentiated cells. In addition, the polypeptides inhibit the differentiation of myoblasts into myotubes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/725,876, filed Dec. 1, 2003, which is a divisional of U.S.application Ser. No. 09/684,579, filed Oct. 6, 2000, issued as U.S. Pat.No. 6,670,450, which is a continuation-in-part of PCT/JP99/01913, filedApr. 9, 1999, which claims priority from Japanese Application 10/115975,filed Apr. 10, 1998. The disclosures of all of the above applicationsare incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to a novel protein involved in myocytedifferentiation and DNA encoding the protein.

BACKGROUND OF THE INVENTION

Genes, such as muscle creatine kinase, troponin, caveolin 3, α-actin,and myosin, are reported to be expressed predominantly in the skeletalmuscles. A family of transcription factors specifically expressed in themuscles, including myoD, myogenin, myf-5, and MRF-4/herculin/myf-6, havebeen cloned. These factors are phosphorylated nuclear proteinscontaining a helix-loop-helix (bHLH) motif, as required for bothdimerization and DNA binding, and are believed to be determinants of thecell-specific differentiation program (Olson and Klein (1994), Genes &Dev. 8:1-8). When one of these factors is introduced into non-myogeniccells, differentiation into mature muscle cells is initiated (Weintraubet al. (1991), Science 251:761-766). The myoD family, a group oftranscription factors, has been found to direct muscle formation,inhibit proliferation, activate differentiation and induce a contractilephenotype. While myoD and myf-5 are expressed within the proliferatingmyoblasts, myogenin and MRF-4 are not expressed until the myoblastswithdraw from the cell cycle in response to mitogen withdrawal. Based onthese findings, it was demonstrated that myogenin and MRF-4 activate andmaintain the expression of muscle-specific genes (Emerson (1993), Curr.Opin. Genet. Dev. 3:265-274), while myoD and myf-5 are thought to play arole in the proliferation of myoblasts. Other cell-cycle regulatoryproteins, such as RB (Shiio et al. (1996), Oncogene 12:1837-1845, Wanget al. (1997), Cancer Research 57:351-354), p21 (Guo et al. (1995), Mol.Cell Biol. 15:3823-3829), cyclin D, cdk2, cdk4 (Kiess et al. (1995),Oncogene 10:159-166) and tumor suppressor gene p53 (Soddu et al. (1996),J. Cell Biol. 134:193-204) are involved in the muscle celldifferentiation program. Recently, caveolin 3 (Song et al. (1996), J.Cell Biol. 271:15160-15165), α-dystroglycan (Kostrominova and Tanzer(1995), J. Cell Biochem. 58:527-534) and DNA methyltransferases (Takagiet al. (1995), Eur. J. Biochem. 231:282-291) have been shown to playpositive roles in myogenic differentiation.

SUMMARY OF THE INVENTION

An objective of the present invention is to provide a novel protein andgene involved in myocyte differentiation, and the production and usethereof.

The inventors carried out an antibody screening, using an antibodyraised against a protein specific to immortalized cells, to isolategenes specifically expressed in the immortalized cells. Unexpectedly, anovel gene was isolated, which was not an initial objective gene. Byanalyzing the isolated gene, the inventors found that this gene is anovel gene showing no significant homology with any known genesdeposited in the database, and is strongly expressed in skeletal muscleand undifferentiated cells. The inventors also analyzed the proteinencoded by the gene, and found that the protein has an inhibitory effecton the differentiation of myoblasts into myotubes. The inventors alsofound that the protein interacts with p53, a transcription factorinvolved in tumor suppression, to inhibit the p53 transactivationfunction.

The present invention relates to a novel protein having an inhibitoryeffect on the differentiation of myoblasts into myotubes, and the geneencoding the protein, and the production and the use thereof. Morespecifically the present invention relates to:

(1) a protein comprising the amino acid sequence of SEQ ID NO:1, or aprotein comprising said amino acid sequence in which one or more aminoacids are substituted, deleted or added and exhibiting an inhibitoryeffect on the differentiation of myoblasts into myotubes;

(2) a protein encoded by DNA that hybridizes with the DNA comprising thenucleotide sequence of SEQ ID NO:2, wherein said protein exhibits aninhibitory effect on the differentiation of myoblasts into myotubes;

(3) a DNA encoding the protein according to (1);

(4) a DNA hybridizing with the DNA comprising the nucleotide sequence ofSEQ ID NO:2, wherein said DNA encodes a protein exhibiting an inhibitoryeffect on the differentiation of myoblasts into myotubes;

(5) a vector containing the DNA according to (3);

(6) a transformant retaining the DNA according to (3) in an expressiblemanner;

(7) a method for producing the protein according to (1) or (2), saidmethod comprising culturing the transformant according to (6);

(8) an antibody binding to the protein according to (1) or (2);

(9) a method of screening for a compound that binds to the proteinaccording to (1) or (2), said method comprising the steps of:

a) contacting a test sample with said protein or a partial peptidethereof;

b) detecting the binding activity of the test sample to said protein ora partial peptide thereof; and

c) selecting a compound binding to said protein or a partial peptidethereof;

(10) a compound, binding to the protein according to (1) or (2), whereinsaid compound can be isolated using the method according to (9);

(11) a method of screening for a compound that promotes or inhibits theactivity of the protein according to (1) or (2), the method comprisingthe steps of:

a) contacting myoblasts with said protein in the presence of a testsample;

b) detecting the differentiation of the cells into myotubes; and

c) selecting a compound which can increase or decrease the inhibitoryactivity of the protein, compared with its inhibitory activity asdetected in the absence of said test sample;

(12) a method of screening for a compound that promotes or inhibits theactivity of the protein according to (1) or (2), said method comprisingthe steps of:

a) providing p53-deficient cells with a vector expressing said protein,a vector expressing p53, and a vector expressing a reporter gene inresponse to p53;

b) contacting a test sample with said cells;

c) detecting the reporter activity in said cells; and

d) selecting a compound that can reduce or increase the reporteractivity compared with the activity in the cells without contact withsaid test sample (control);

(13) a compound that promotes or inhibits the activity of the proteinaccording to (1) or (2), wherein said compound can be isolated using themethod according to (11) or (12); and

(14) a DNA comprising at least 15 nucleotides in length and specificallyhybridizing with the DNA comprising the nucleotide sequence of SEQ IDNO:2.

The present invention relates to a novel protein, “striamin,” thatinhibits the differentiation of myoblasts into myotubes. (The inventorsinitially designated the protein “striatin” in the original application(Japanese Patent Application No. Hei 10-115975), but another protein waslater found to have the same name; hence the renaming to “striamin”).The nucleotide sequence of striamin cDNA derived from mouse DNA is shownin SEQ ID NO:1, and the amino acid sequence of the protein encoded bythe cDNA is shown in SEQ ID NO:2. As shown in SEQ ID NO:1, mousestriamin cDNA has an ORF encoding a protein of 149 amino acids. Asdetermined by immunoprecipitation of the striamin protein translated invitro (FIG. 2A), and by Western blotting of the recombinant striaminprotein (FIG. 2B), the mouse-derived striamin protein has a molecularweight of about 18 kDa. Northern blot analysis showed that the striamingene is expressed in the undifferentiated cells, and that the expressionof this gene is inhibited during myoblast differentiation into myotubes(FIG. 4C). Overexpression of the gene actually blocked thedifferentiation of myoblasts into myotubes (FIG. 5). These facts suggestthat the striamin protein is involved in the duration of theundifferentiated state of the cells.

The expression of the striamin protein also inhibited expression of thep53 transactivation function. The expression of this transcriptionfactor is known to be upregulated during muscle differentiation (FIG. 7and FIG. 8). The striamin protein was further shown to interact with p53both in vivo and in vitro (FIG. 9 and FIG. 10). It is reported that p53activity increases substantially in the process of muscle formation invitro. Inhibition of p53 activity by striamin is quite consistent withthe down-regulation of striamin during myogenesis. These facts suggestthat striamin affects muscle formation through direct interaction withp53.

The striamin protein of the present invention may be prepared as arecombinant protein by making use of recombinant technology, and as anaturally occurring protein. For example, a recombinant protein can beprepared, as described below, by culturing cells transformed with DNAencoding the striamin protein. In addition, a naturally occurringprotein can be isolated from tissues, such as skeletal muscles, usingmethods known by a person skilled in the art, for example, by performingaffinity chromatography using an antibody that binds to the striaminprotein as described below. The antibody may be a polyclonal ormonoclonal antibody. Polyclonal antibodies can be prepared, for example,by obtaining serum from a small animal, such as a rabbit, that isimmunized with the striamin protein, followed by purification using, forexample, ammonium sulfate precipitation, protein A column, protein Gcolumn, DEAE ion exchange chromatography, and striamin protein coupledaffinity column. Monoclonal antibodies can be prepared as follows. Asmall animal, such as a mouse, is immunized with the striamin protein.The mouse is then dissected to remove the spleen, which is subsequentlyhomogenized to dissociated cells. These are then fused to mouse myelomacells using a reagent such as polyethylene glycol, and the fused cells(hybridomas) thus obtained are subjected to the selection of clonesproducing antibodies against the protein. Subsequently, a hybridoma cellthus obtained is transplanted into a mouse intraperitoneally, and theascites fluid is recovered from the mouse, followed by purificationusing, for example, ammonium sulfate precipitation, protein A column,protein G column, DEAE ion exchange chromatography, and striamin proteincoupled affinity column.

When the antibody obtained is for human use (e.g., for antibodytherapy), a humanized or human antibody is advantageous to reduce theimmunogenicity. Among methods for humanizing antibodies, the CDRgrafting method is well known. In this method, the antibody gene iscloned from the cell producing the monoclonal antibody and its antigendeterminant portion is grafted to an existing human antibody.Alternatively, human antibodies can be prepared directly, by the samemethod used for conventional monoclonal antibodies, i.e., by immunizinga mouse whose immune system is replaced with a human's immune system.

In addition, as well as preparing a native striamin protein, one skilledin the art can prepare modified proteins whose functions are equivalentto those of the native protein (e.g., an inhibitory effect on thedifferentiation of myoblasts into myotubes, binding activity to p53, aninhibitory effect on the p53 transactivation function), using awell-known method for modifying proteins, such as for substitution ofamino acid residues in the protein. Spontaneous mutation of an aminoacid in the protein may possibly occur. Thus, the proteins of thepresent invention include mutant proteins whose amino acid sequencesdiffer from that of the native protein by amino acid substitution,deletion and/or addition, and whose function is equivalent to the nativeprotein. The methods for modifying amino acids, which are well-known toone skilled in the art, include the site-directed mutagenesis system byPCR (GIBCO-BRL, Gaithersburg, Md.), the site-directed mutagenesis methodusing oligo-nucleotides (Kramer, W. and Fritz, H. J. (1987), Methods inEnzymol., 154:350-367), and Kunkel's method (Methods in Enzymol. (1988),85:2763-2766). Amino acid substitutions are made at typically 10 or lessresidues, preferably six or less residues and more preferably three orless residues.

A “conservative amino acid substitution” is one in which an amino acidresidue is replaced with another residue having a chemically similarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

Any inhibitory effect of the proteins thus prepared on thedifferentiation of myoblasts into myotubes can be detected using, forexample, a method such as that for determining differentiation potencyby using cultured mouse C2C12 myoblast cell line. (When cultured inserum-free DMEM medium or DMEM medium containing 2% equine serum, mouseC2C12 myoblast cell line is differentiated into multinucleate myotubecells.) In this method, a C2C12 myoblast cell line is cultured in thepresence of a test protein to determine the potency of thedifferentiation into multinucleate myotube cells (See Example 6).Binding of the prepared protein to p53 can be detected, for example, bycontacting the two proteins in vitro or in vivo, which are thensubjected to immunoprecipitation with an anti-p53 antibody, an antibodyagainst the prepared protein or, if a tag is added to either protein, anantibody against the tag, and subsequently by Western blotting (seeExamples 10 and 11). Inhibition of p53 transactivation by the preparedprotein can be detected, for example, by determining the reporteractivity of the cells into which both a vector expressing p53 and avector carrying a p53-responsive reporter, and subsequently a vectorexpressing the prepared protein, are introduced. Reporter activity isthen compared with that of a control, i.e., cells not harboring thevector expressing the prepared protein (See Examples 8 and 9).

It is also well within the art of a person with ordinary skill to obtaina protein functionally equivalent to the mouse striamin protein (SEQ IDNO:1) by isolating DNA showing significant homology with the DNA thatencodes the mouse striamin protein (SEQ ID NO:2) or a part thereof,using technology such as a hybridization technique (Sambrook et al.,Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. Press,1989). Thus, the proteins of the present invention also include thoseproteins that are encoded by DNA hybridizing with the DNA encoding themouse striamin protein, and that are functionally equivalent to themouse striamin protein (e.g., the protein that was detected in Example5, which is encoded by a human transcript 3.1 kb in length). Whenhybridizing DNA is isolated from other organisms, animals including, butnot limited to, humans, rats, rabbits, and cattle are used for theisolation. For this purpose, tissues such as skeletal muscles, inparticular, are suitable. DNAs thus isolated, which encode proteinsfunctionally equivalent to the mouse striamin protein, generally showsignificant homology with the DNA (SEQ ID NO:2) encoding the mousestriamin protein (SEQ ID NO:1). The term “significant homology”indicates a sequence identity of at least 40%, preferably at least 60%,more preferably at least 80%, and most preferably at least 95% at aminoacid level. The degree of homology can be determined according to thealgorithm described in the literature (Wilbur, W. J. and Lipman, D. J.Proc. Natl. Acad. Sci. USA (1983), 80:726-730).

The “percent identity” of two amino acid sequences or of two nucleicacids is determined using the algorithm of Karlin and Altschul (Proc.Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin andAltschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotidesearches are performed with the NBLAST program, score=100,wordlength=12. BLAST protein searches are performed with the XBLASTprogram, score=50, wordlength=3. Where gaps exist between two sequences,Gapped BLAST is utilized as described in Altschul et al. (Nucleic AcidsRes. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are used. The programs are available at the web siteof the National Center for Biotechnology Information.

Examples of conditions used for the hybridization are as follows. For“low stringency” hybridization, after prehybridization for at least 30minutes at 55° C. using “ExpressHyb Hybridization Solution” (CLONTECH),hybridization is carried out by adding a labeled probe and incubatingfor at least one hour at 37 to 55° C., followed by washing the filterthree times in 2×SSC containing 0.1% SDS for 20 minutes at roomtemperature and then once in 1×SSC containing 0.1% SDS for 20 minutes at37° C. For “medium stringency” hybridization, after prehybridization forat least 30 minutes at 60° C. using “ExpressHyb Hybridization Solution”(CLONTECH), hybridization is carried out by adding a labeled probe andincubating for at least one hour at 60° C., followed by washing thefilter three times in 2×SSC containing 0.1% SDS for 20 minutes at roomtemperature and then twice in 1×SSC containing 0.1% SDS for 20 minutesat 50° C. For “high stringency” hybridization, after prehybridizationfor at least 30 minutes at 68° C. using “ExpressHyb HybridizationSolution” (CLONTECH), hybridization is carried out by adding a labeledprobe and incubating for at least one hour at 68° C., followed bywashing the filter three times in 2×SSC containing 0.1% SDS for 20minutes at room temperature and then twice in 0.1×SSC containing 0.1%SDS for 20 minutes at 50° C.

The present invention also relates to the DNA encoding the striaminprotein of the present invention. The DNA of the present invention maybe any DNA including genomic DNA and synthetic DNA as well as cDNA, aslong as it encodes the aforementioned striamin protein. The DNA of thepresent invention can be used, for example, to produce recombinantproteins. Such recombinant proteins can be prepared by inserting the DNAof the present invention (e.g., SEQ ID NO:1) into an appropriateexpression vector. This is then introduced into appropriate cells toobtain a transformant, followed by culturing the transformants, and bypurifying the expressed protein. Cells used for the production ofrecombinant proteins include, but are not limited to, mammalian cellssuch as COS, CHO, and NIH3T3 cells, insect cells such as Sf9 cells,yeast cells, and E. coli cells. Although vectors used for expression ofrecombinant proteins in the cell will vary depending on the host cells,such vectors include, for example, pcDNA3 (Invitrogen) and pEF-BOS(Nucleic Acids Res. 1990, 18 (17), p. 5322) for expression in mammaliancells, “BAC-to-BAC baculovirus expression system” (GIBCO BRL) forexpression in insect cells, “Pichia Expression Kit” (Invitrogen) forexpression in yeast cells, and pGEX-5X-1 (Pharmacia) and “QIAexpresssystem” (Qiagen) for expression in E. coli cells. Introduction of thevector into the host cells can be carried out using methods includingthe calcium phosphate, DEAE dextran, cationic liposome DOTAP (BoehringerMannheim), electroporation, and calcium chloride methods. Thetransformants can be cultured using a method well known to one skilledin the art, using an appropriate existing method, depending on theproperties of the particular transformant. Recombinant proteins may bepurified from the transformants thus obtained may be carried out using,for example, the method described in the literature “TheQiaexpressionist Handbook, Qiagen, Hilden, Germany.”

The DNA of the present invention can be used in gene therapy fordiseases caused by mutations that have occurred in the DNA. As used ingene therapy, the DNA of the present invention is inserted into a vectorsuch as adenoviral (e.g., pAdexLcw) or retroviral (e.g., pZIPneo)vectors for in vivo administration. Administration can be carried outvia either an ex vivo or in vivo process.

The present invention also features a DNA molecule that contains atleast 15 nucleotides, and that can specifically hybridize with the DNAencoding the striamin protein of the present invention or thecomplementary DNA thereof. The term “specifically hybridize” indicatesthat no significant cross-hybridization occurs to DNA encoding otherproteins under standard hybridization conditions, preferably understringent hybridization conditions. Such DNA molecules include probes,primers and nucleotides, and nucleotide derivatives (e.g., antisenseoligonucleotides ribozymes, etc.), that can specifically hybridize tothe DNA encoding the protein of the present invention or to thecomplementary DNA thereof.

An “isolated nucleic acid” is a nucleic acid the structure of which isnot identical to that of any naturally occurring nucleic acid or to thatof any fragment of a naturally occurring genomic nucleic acid spanningmore than three separate genes. The term therefore covers, for example,(a) a DNA which has the sequence of part of a naturally occurringgenomic DNA molecule but is not flanked by both of the coding sequencesthat flank that part of the molecule in the genome of the organism inwhich it naturally occurs; (b) a nucleic acid incorporated into a vectoror into the genomic DNA of a prokaryote or eukaryote in a manner suchthat the resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein.Specifically excluded from this definition are nucleic acids present inmixtures of different (i) DNA molecules, (ii) transfected cells, or(iii) cell clones: e.g., as these occur in a DNA library such as a cDNAor genomic DNA library.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. The substantially pure polypeptide is atleast 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Puritycan be measured by any appropriate standard method, for example, bycolumn chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

The present invention includes an antisense oligonucleotide hybridizingto any portion of the nucleotide sequence of, for example, SEQ ID NO:2.Preferably, such an oligonucleotide is antisense to a continuous 15nucleotides or more in length in the nucleotide sequence of SEQ ID NO:2.More preferably, the aforementioned continuous sequence 15 nucleotidesor more in length contains a translation initiation codon.

Derivatives or modified oligonucleotides can be used as an antisenseoligonucleotide. Such modified nucleotides include loweralkylphosphonate-modified, such as methylphosphonate- orethylphosphonate-modified, phosphorothioate-modified, andphosphoroamidate-modified nucleotides.

As used here, the term “antisense oligonucleotides” means not only anoligonucleotide complementary to all of the continuous nucleotidescomprising the given region of DNA or mRNA, but also oligonucleotideshaving one or more nucleotides mismatched against the continuousnucleotides, as long as DNA or mRNA and the oligonucleotides are able tospecifically hybridize to the nucleotide sequence of SEQ ID NO:2.

Such DNAs include continuous nucleotide sequences at least 15nucleotides in length, showing at least 70%, preferably at least 80%,more preferably at least 90%, and most preferably at least 95% homologywith the nucleotide sequence of SEQ ID NO:2. An algorithm that can beused to determine the extent of homology is given herein. These DNAs areuseful as probes to detect or isolate the DNA encoding the protein ofthe present invention, according to the methods described below in theExamples. They are also useful primers for amplification.

The antisense oligonucleotide derivatives of the present invention actupon the cells producing the protein of the present invention. They bindto the DNA or mRNA encoding the protein, inhibiting the transcription ortranslation of the protein and promoting the degradation of the mRNA. Asexpression of the protein is inhibited, there is an inhibitory effect onthe functioning of the protein.

The antisense oligonucleotide derivatives can be formulated intoexternal preparations such as liniments and poultices by mixing with asuitable base material, which is inert to the derivatives.

If necessary, the derivatives can be formulated into tablets, powders,granules, capsules, liposome capsules, injections, solutions, nose-dropsand into freeze-dried agents, by adding excipients, isotonic agents,dissolving auxiliaries, stabilizers, preservatives and pain-killers.These formulations can be prepared using a standard technique.

An antisense oligonucleotide derivative of the present invention couldbe given to a patient by direct application onto the affected site or byintravascular administration. A mounting medium for including antisensederivatives can also be used to increase sustainability andmembrane-permeability of the formulations. For example, liposome, poly-Llysine, lipid, cholesterol and lipofectin or derivatives thereof can beused.

A range of dosages of the antisense oligonucleotide derivatives, from0.1 to 100 mg/kg, can be administered, depending on the patients'conditions.

The antisense oligonucleotides, or an inhibitor containing an antisenseoligonucleotide, inhibit the expression of the protein of the presentinvention, and are thus useful in inhibiting its biological activity.

The present invention also features a screening method for a compoundthat binds to the protein. The screening method comprises the followingsteps of:

(a) contacting a test sample with the protein of the present inventionor a partial peptide thereof;

(b) detecting the binding activity of the test sample to the protein ofthe present invention or a partial peptide thereof; and

(c) selecting a compound binding to the protein of the present inventionor a partial peptide thereof.

The protein of the present invention used for screening may berecombinant or naturally occurring protein, or may be a partial peptidethereof. Any test sample can be used without particular restriction,including, for example, cell extracts, culture supernatants, productsfrom fermented microorganisms, extracts from marine organisms, plantextracts, purified or crude proteins, peptides, nonpeptidic compounds,synthetic low molecular weight compounds and natural compounds.

A number of methods well-known to one skilled in the art can be used toscreen for a protein binding to the protein of the present inventionutilizing the protein of the present invention. One of these screeningmethods is immunoprecipitation. Typically, immunoprecipitation isconducted as follows. The gene encoding the protein of the presentinvention is inserted downstream of a promoter provided for expressingforeign genes, such as pSV2neo, pcDNA 1, and pCD8, to express the gene,for example, in mammalian cells. Any commonly available promoter may beused for the expression, including SV 40 early promoter (Rigby inWilliamson (ed.), Genetic Engineering Vol. 3, Academic Press, London, p.83-141 (1982)), EF-1α promoter (Kim et al., Gene, 91:217-223 (1990)),CAG promoter (Niwa et al., Gene, 108:193-200 (1991)), RSV LTR promoter(Cullen, Methods in Enzymology, 152:684-704 (1987)), SR a promoter(Takebe et al., Mol. Cell. Biol. 8:466 (1988)), CMV immediate earlypromoter (Seed and Aruffo, Proc. Natl. Acad. Sci. USA, 84: 3365-3369(1987)), SV 40 late promoter (Gheysen and Fiers, J. Mol. Appl. Genet.,1:385-394 (1982)), and Adenovirus late promoter (Kaufman et al., Mol.Cell. Biol., 9:946 (1989), HSV TK promoter.

Any method for introducing and expressing a foreign gene in animal cellsmay be used to express the gene, including electroporation (Chu, G. etal., Nucl. Acid. Res. 15:1311-1326 (1987)), a calcium phosphate method(Chen, C. and Okayama, H. Mol. Cell. Biol. 7:2745-2752 (1987)), a DEAEdextran method (Lopata, M. A. et al., Nucl. Acid. Res. 12: 5707-5717(1984); Sussman, D. J. and Milman, G., Mol. Cell. Biol., 4:1642-1643(1985)), Lipofectin method (Derijard, B., Cell, 7:1025-1037 (1994);Lamb, B. T. et al., Nature Genetics, 5:22-30 (1993); Rabindran, S. K. etal., Science, 259:230-234 (1993)).

The protein of the present invention can be expressed as a fusionprotein containing a monoclonal antibody recognition site, thespecificity of which has been defined by introducing the monoclonalantibody recognition site (epitope) at the N or C terminus of theprotein of the present invention. Epitope-antibody systems used for thispurpose are commercially available (Jikken Igaku, Experimental Medicine,13:85-90 (1995)). A number of vectors provided for expression of a geneas a fusion protein fused with β-galactosidase, a maltose bindingprotein, glutathione S-transferase, or a green fluorescent protein (GFP)via a multi-cloning site available from commercial sources.

To limit variation in characteristics between the protein of the presentinvention and its fusion protein, an epitope of restricted size isintroduced to prepare the fusion protein. This can range from just belowto just above ten amino acids. Such methods have been reported. Acombination of an epitope, such as polyhistidine (His-tag), influenzahemagglutinin HA, human c-myc, FLAG, Vesicular stomatitis viralglycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human herpes simplexviral glycoprotein (HSV-tag) or E-tag (epitopes on monoclonal phages),and a monoclonal antibody that recognizes the epitope can be used as anepitope-antibody system to screen for a protein binding to the proteinof the present invention (Jikken Igaku (1995), Experimental Medicine,13:85-90).

In immunoprecipitation, an immunocomplex is formed when these antibodiesare added to the cell lysate prepared using appropriate detergent. Theimmunocomplex comprises the protein of the present invention, a proteincapable of binding to it, and the antibody. In addition to using theantibody against the epitope mentioned above, an antibody raised againstthe protein of the present invention can be used for theimmunoprecipitation. Antibodies against the protein of the presentinvention can be prepared, for example, by introducing the gene encodingthe protein of the present invention into an appropriate E. coliexpression vector to express the protein in E. coli cells, purifying theexpressed product, and immunizing animals, such as rabbits, mice, goats,and chickens, with the purified protein. Alternatively, the antibodiesagainst the protein of the present invention can be prepared byimmunizing the above animals with synthetic partial peptides of theprotein of the present invention.

Immunocomplexes can be precipitated, for example, by using Protein ASepharose or Protein G Sepharose, if the antibody is a mouse IgGantibody. When the protein of the present invention is prepared asfusion proteins fused with epitopes such as GST, substances specificallybinding to the epitopes, such as glutathione-Sepharose 4B, can be usedto form the immunocomplexes in the way that an antibody against theprotein of the present invention is used.

A standard method of immunoprecipitation may be carried out as describedin the literature; for example, Harlow, E and Lane, D: Antibodies, pp.511-552, Cold Spring Harbor Laboratory publications, New York (1988).

SDS-PAGE is typically used to analyze the immunoprecipitated proteins.By using a gel with appropriate density, the bound proteins can beresolved by molecular size. In this procedure, the cells are cultured ina medium containing a radioisotope, such as 35S-methionine or35S-cysteine, to label the proteins in the cells. The detectionsensitivity is thereby increased, since, in general, proteins bound tothe protein of the present invention are difficult to detect usingconventional protein-staining methods such as Coomassie and silverstaining. Once the molecular size of the protein is clarified, theprotein of interest can be directly purified from SDS-polyacrylamide geland subjected to sequencing.

West-Western blotting (Skolnik, E. Y. et al. Cell (1991), 65:83-90), forexample, may be used to isolate proteins binding to the protein of thepresent invention by using the protein. In this method, the isolation iscarried out by constructing a cDNA library from cells, tissue or anorgan presumed to express binding proteins of the protein of the presentinvention (for example, myoblast cells and NIH3T3 cells) using a phagevector (e.g., λgt11, ZAP), expressing the vectors on LB-agarose, fixingthe expressed proteins on the filter, reacting the filter with thelabeled and purified protein of the present invention, and detecting theplaques expressing the proteins bound to the protein of the presentinvention through the label. The methods of labeling the protein of thepresent invention include those utilizing the affinity between biotinand avidin; those using an antibody binding specifically to the proteinof the present invention, or a peptide or polypeptide fused to theprotein of the present invention (for example, GST); those using aradioisotope; and those using fluorescence.

Another screening method of the present invention is to use thetwo-hybrid system using cells (Fields, S., and Sternglanz, R., Trends.Genet. (1994), 10:286-292). In two-hybrid systems methods, such as“MATCHMAKER Two-hybrid System,” “Mammalian MATCHMAKER Two-hybrid AssayKit,” and “MATCHMAKER One-Hybrid System” (Clontech), or “HybriZAPTwo-Hybrid Vector System” (Stratagene), and also as described in‘Characterization of SAP-1, a protein recruited by serum response factorto the c-fos serum response element’ (Dalton S and Treisman R (1992),Cell 68:597-612), the protein of the present invention is fused to theSRF binding region or GAL4 binding region and expressed in yeast cells.

A cDNA library was prepared from cells predicted to express a proteinthat will bind to the protein of the present invention so as to expressproteins fused to the VP16 or GAL4 transcriptional activation region.The cDNA library is then introduced into the above yeast cells, and thecDNA derived from the library is isolated from the positive clonesdetected (when a protein binding to the protein of the present inventionis expressed in yeast cells, the binding of the two activates a reportergene making positive clones detectable). The isolated cDNA can beintroduced into E. coli to express a protein encoded by it. This methodalso allows preparation of a protein binding to the protein of thepresent invention and the gene encoding it. The reporter genes includeAde2, LacZ, CAT, and luciferase genes as well as HIS3 gene.

Affinity chromatography can be used to screen for the compound bindingto the protein of the present invention. For instance, the protein ofthe present invention is immobilized on the support of the affinitycolumn, to which a test sample is applied. The test sample is selectedto express the protein that binds to the protein of the presentinvention; a cell extract or cell lysate can be used. After the testsample is applied, the column may be washed to prepare the protein boundto the protein of the present invention.

The amino acid sequence of the protein thus obtained may be analyzedand, based on the result, oligo-DNA is synthesized and used as a probefor screening the cDNA library to obtain the DNA encoding the protein.

The present invention may include use of a biosensor in which a surfaceplasmon resonance phenomenon is utilized to detect or determine thebound compounds. The biosensors utilizing a surface plasmon resonancephenomenon (e.g., BIAcore, Pharmacia) enable the real-time observationof the interaction between the protein of the present invention and thetest compound as surface plasmon resonance signals, using a small amountof the protein without the need for labeling. Consequently, the bindingof the protein of the present invention and the test compound can beestimated using a biosensor such as BIAcore.

Among methods for isolating compounds, not limited to proteins, thatbind to the protein of the present invention, those methods of screeningfor molecules that bind to the protein of the present invention bymaking synthetic compounds, a natural substance bank, or a random phagepeptide display library, act on the immobilized protein of theinvention. Furthermore, methods of screening using a high-throughputbased on combinatorial chemistry techniques are well known to oneskilled in the art. (Wrighton N C; Farrell F X; Chang R; Kashyap A K;Barbone F P; Mulcahy L S; Johnson D L; Barrett R W; Jolliffe L K; DowerW J. ‘Small peptides as potent mimetics of the protein hormoneerythropoietin’, Science (United States) Jul. 26, 1996 273:458-64,Verdine G L., ‘The combinatorial chemistry of nature’. Nature (England)Nov. 7, 1996, 384:11-13, Hogan J C Jr., ‘Directed combinatorialchemistry’. Nature (England) Nov. 7, 1996, 384:17-9).

The present invention also relates to a method for screening for acompound able to promote or inhibit the activity of the protein of theinvention. Since the protein of the present invention has inhibitoryactivity on the differentiation of myoblasts into myotubes, a compoundable to promote or inhibit activity of the protein of the invention canbe screened by using this activity as an indicator. Such screening canbe done using a method comprising the steps of:

(a) exposing the protein of the present invention to myoblast cells inthe presence of a test sample,

(b) detecting the differentiation of the cells into myotube cells, and

(c) selecting a compound that increases or reduces the inhibitory effectof the protein of the present invention by comparing with the results ofthe assay performed in the absence of the test sample.

The protein of the present invention used for the screening can be anaturally occurring protein or a recombinant protein, or a purifiedprotein or the supernatant of cell culture (when the protein is secretedfrom the cell).

There are no particular restrictions as to the test samples used. Forexample, cell extracts, culture supernatants, products from fermentedmicroorganisms, extracts from marine organisms, plant extracts, purifiedor crude proteins, peptides, nonpeptidic compounds, synthetic lowmolecular weight compounds and natural compounds may be used.Alternatively, a compound obtained by the aforementioned screening forcompounds binding to the protein of the present invention can be used asa test sample.

Myoblasts used for detecting the differentiation into myotube cells,preferably include, but are not limited to, C2C12 myoblasts. Thedifferentiation into myotubes can be detected by a method, such as thatfor determining the differentiation potency of the C2C12 myoblast cellline into multinucleate myotube cells when they are cultured in thepresence of both the test sample and the protein of the presentinvention, using the culture system of the mouse C2C12 myoblast linedescribed in the Examples (the mouse myoblast cell line C2C12differentiates into multinucleate myotube cells when cultured in theDMEM medium free of serum or containing 2% equine serum).

The protein of the present invention has here been shown to inhibit thetranscriptional factor activity of p53. Therefore, a compound promotingor inhibiting the activity of the protein can also be screened conductedusing the transcriptional factor activity as an indicator. Thisscreening can be carried out by:

(a) providing p53-deficient cells in which vectors expressing theprotein, p53 and a reporter gene responsive to p53 have been introduced,

(b) exposing the test sample to the cells,

(c) detecting the reporter activity in the cells, and

(d) selecting a compound that increases or reduces the reporter activitycompared with the activity detected in the absence of the test sample(control).

Specifically, a test sample is added to the detection system describedin Examples 8 and 9, in which inhibition of the transcriptional factoractivity of p53 by striamin is detected, reporter activity is detected,and then the compound altering the activity may be selected. There areno particular restrictions as to the test samples used. For example,cell extracts, culture supernatants, products from fermentedmicroorganisms, extracts from marine organisms, plant extracts, purifiedor crude proteins, peptides, nonpeptidic compounds, synthetic lowmolecular weight compounds, and natural compounds may be used.Alternatively, a compound obtained by the aforementioned screening forcompounds binding to the protein of the present invention can be used asa test sample.

The striamin expression vector may express striamin fully or it mayexpress a partial peptide of striamin which can inhibit thetranscriptional factor activity of p53 (e.g., C-terminus of striamin).Preferably, the chosen p53 vector should express p53 controllably. Suchvectors include temperature-sensitive p53 expression vectors.

A plasmid expressing the reporter in response to p53 has the reportergene located downstream of a p53 responsive sequence. “ATGCTTGCCC” maybe used as the p53 responsive sequence. There are no particularrestrictions as to the reporter gene used as long as it has a detectableresponse. Genes such as those for luciferase and β-galactosidase can beused.

Preferably, p53-deficient cells are used in vector introduction to avoidthe expression of the reporter gene by the action of endogenous p53.Such cells include p53−/−murine fetal fibroblast cells.

If a reduction in reporter activity is detected during screening,compared with the activity found in the absence of the test sample(control), then the test sample used is determined as being a compound(or containing a compound) that promotes the activity of the protein ofthe present invention. Likewise, when the test sample increases thereporter activity, it is determined as being a compound (or containing acompound) that inhibits the activity of the protein of the presentinvention.

As used in the screening method, “a compound that promotes or inhibitsthe activity of the protein of the present invention” is any compound,without limit, promoting or inhibiting the signal transduction via theprotein of the present invention. Specifically, such a compound is notlimited to ones that promote or inhibit the activity by directly actingon the protein of the present invention. A compound that promotes orinhibits the signal transduction by action on any of the downstreamfactors from the protein of the present invention in the signaltransduction is also included.

The compounds obtained from the screening of the present invention willbe candidate agents that promote or inhibit the activity of the proteinof the present invention, or that promote or inhibit the signaltransduction via the protein of the present invention, for a diseaseassociated with the protein of the present invention. The compoundsobtained using the screening method of the present invention include anysubstance in which a portion of the structure of a compound having thebinding activity to the protein of the present invention that isobtained using the screening method of the present invention and hasbeen altered by addition, deletion and/or substitution.

To use the compounds obtained with the screening method of the presentinvention as drugs for humans and other mammals such as mice, rats,guinea pigs, rabbits, chickens, cats, dogs, sheep, pigs, cattle,monkeys, baboons, and chimpanzees, the compounds may be formulated foradministration to the patients by a well-known pharmaceuticalpreparation method, as well as direct administration of the isolatedcompounds. For example, the drugs can be administered orally in the formof sugar-coated tablets, capsules, elixirs, or microcapsules, orparenterally in the form of injections of sterile solutions orsuspensions with water or any other pharmaceutically acceptable liquidas needed. The compounds may be formulated by adequately combining themwith pharmacologically acceptable carriers or media, specifically,sterilized water or physiological saline, vegetable oil, emulsifiers,suspensions, surfactants, stabilizers, flavoring agents, excipients,vehicles, preservatives and bonding agents, and mixing in a unit doseform required for generally accepted drug implementation. The amount ofeach effective ingredient in these preparations is provided for giving asuitable dosage acquirable within the indicated range.

Additives mixable in the tablets and capsules include a bonding agentsuch as gelatin, corn starch, tragacanth gum or arabic gum; an excipientsuch as crystalline cellulose; a swelling agent such as corn starch,gelatin or alginic acid; a lubricator such as magnesium stearate; asweetener such as sucrose, lactose or saccharin; and a flavoring agentsuch as peppermint, Gaultheria adenothrix oil and cherry. When the unitdosage form is a capsule, a liquid carrier such as oil can also beincluded in the above ingredients. Sterile compositions for injectionscan be formulated following normal drug implementations using vehiclessuch as distilled water used for injections.

Aqueous solutions for injection include physiological saline, andisotonic liquids containing glucose or other adjuvants, such asD-sorbitol, D-mannose, D-mannitol, and sodium chloride. These solutionscan be used in conjunction with suitable dissolution adjuvants, such asalcohol, specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, and non-ionic surfactants such as poly sorbate 80(TM) and HCO-50. Sesame oil or Soy-bean oil can be used as an oleaginousliquid and may be used in conjunction with benzyl benzoate or benzylalcohol as dissolution adjuvants. They may also be formulated with abuffer, such as phosphate and sodium acetate buffer; a pain-killer, suchas procaine hydrochloride; a stabilizer such as benzyl alcohol, phenol;and an anti-oxidant. The injection solution thus prepared is filled intoa suitable ampoule.

Administration to the patients may be carried out using a method wellknown to one skilled in the art, for example, via intranasal, bronchial,intramuscular or oral route, as well as intra-arterial, intravenous orsubcutaneous injection. While the doses vary depending on the patient'sbody weight and age and on the administration method, one skilled in theart may properly determine the adequate doses. If the compound can beencoded by DNA, gene therapy may possibly be carried out byincorporating the DNA into a vector for gene therapy. While the doses ofthe drug and the method for administration may vary depending on thepatient's body weight, age and condition, one skilled in the art maydetermine them properly. The administered dose of a compound that bindsto the protein of the present invention or promotes or inhibits theactivity of the protein of the present invention will vary depending onthe patient's condition. For oral administration, about 0.1 to about 100mg per day, preferably about 1.0 to 50 mg per day, and more preferablyabout 1.0 to 20 mg per day may typically be administered to a normaladult (weighing 60 kg).

The parenteral dose to be administered varies depending on the subjectfor administration, target organ, subject's conditions, and the methodof administration. In the form of an injection, for example, a dose ofabout 0.01 to about 30 mg per day, preferably about 0.1 to about 20 mgper day, and more preferably about 0.1 to about 10 mg per day may beadvantageously administered to a normal adult (weighing 60 kg) byintravenous injection. For other animals, the dose calculated to bodyweight of 60 kg may be administered to the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of striamin and its predicted aminoacid sequence. The sequence of in-frame codons and the 5′ upstreamsequence obtained by 5′ RACE PCR on the mouse skeletal muscle cDNA areunderlined.

FIG. 2A shows the product from the in vitro translation system ofplasmid pBS/striamin in which striamin ORF is placed downstream of theT3 promoter. This product is detected as a protein of 18 kD (indicatedby an arrowhead) on SDS-PAGE.

FIG. 2B shows the Western blotting of the recombinant striamin proteinobtained from E. coli transformed with IPTG-induced pQE30/striamin,using an anti-His antibody. The signal was detected at the position ofapproximately 18 kD (indicated by an arrow).

FIG. 3 shows the tissue specificity of the striamin expression in mice(A) and humans (B) analyzed by Northern blotting.

FIG. 4A shows detection of the striamin expression in different musclesby Northern analysis. The expression was detected in the fast-twitchfibers (quadriceps, Lane 1), but not in the slow-twitch fibers (soleusmuscle, Lane 2).

FIG. 4B shows the Northern analysis of RNA from the muscle fibersincluding fast- and slow-twitch fibers at various ratios.

Lane 1, quadriceps (95% fast-twitch fibers 2B, 4% fast-twitch fibers2X);

Lane 2, extensor digitorum longus muscle (60% fast-twitch fibers 2B, 28%fast-twitch fibers 2X, 12% fast-twitch fibers 2A);

Lane 3, outer layer of the gastrocnemius muscle (100% fast-twitch fibers2B);

Lane 4, diaphragm (57% fast-twitch fibers 2X, 34% fast-twitch fibers 2A,7% slow-twitch fibers);

Lane 5, soleus muscle (45% fast-twitch fibers 2A, 55% slow-twitchfibers). Striamin is intensively expressed in the fast-twitch fibers 2Bthat have a glycolytic function. 18S ribosomal RNA was used as acontrol.

FIG. 4C shows the striamin expression in the differentiating C2C12 cellsin vitro.

Lane 1 RNA from the myoblasts differentiating in low-density culture.

Lane 2 RNA from the differentiating myoblasts in moderate-density cellculture.

Lane 3 RNA from C2C12 cells cultured for one day in the differentiationmedium;

Lane 4 RNA from C2C12 cells cultured for four days in thedifferentiation medium and forming a large number of myotubes. 18Sribosomal RNA was used as a control.

FIG. 5 shows the effect of striamin on the in vitro differentiation ofC2C 12 cells. Myotube formation was not observed in the cellstransformed with striamin and cultured for 72 hours in thedifferentiation medium (B), but was observed in the control (A).

FIG. 6 depicts the intracellular localization of striamin. In COS7 cellstransfected with pEGFPC1-striamin, green fluorescence from theGFP-striamin fusion protein was identified in the nuclei (A). The nucleishown in A were stained with Hoechst dye (B) and indicated by arrowheads.

To show that their expressions are localized in the nucleus, perinuclearspace, and cytoplasm, pEGFPC1-striamin (a), pEGFPC1-striamin N75 (b),and pEGFPC1-striamin C74 (c) were microinjected into NIH3T3 cells.

FIG. 7 shows the results of the detection of the interaction betweenstriamin and p53 using luciferase as a reporter. The p53 expressionplasmid, the p53-responsive luciferase expression plasmid, and thedifferent striamin expression plasmids were introduced into p53−/−mouseembryonic fibroblast (MEF) cells, and the luciferase activity wasdetected. The amount of each plasmid (0.5 μg/μl) used is indicated bythe FIGS. “1” and “2”. Error bars denote the standard deviations (n=3).

FIG. 8 shows the results of the detection of the interaction betweenstriamin and p53 using β-galactosidase as a reporter. The p53 expressionplasmid, the p53-responsive β-galactosidase expression plasmid, and thedifferent striamin expression plasmids were introduced into p53−/−mouseembryo fibroblast (MEF) cells, and the β-galactosidase activity wasdetected (arrows in d, e, and f). In addition, to identify the cellsinjected with DNA, control IgG was microinjected together with the aboveplasmids into the cells to detect FITC conjugated anti-rabbit IgG (a, b,and c).

Control, a and d; sense-vectors: b and e; antisense-vectors: c and f.FIG. 9 shows the results of detecting the interaction between striaminand p53 in vitro. Different striamins conjugated to histidines werereacted with p53 conjugated to GST in vitro, and the reaction wasimmunoprecipitated with glutathione Sepharose beads, followed by Westernblot analysis using anti-histidine-tag and anti-p53 antibodies (“GST-p53pull” in the figure).

“In put” in the figure represents the results of Western blot analysisof the reaction made without adding GST. “GST-Pull” represents theresults of the immunoprecipitation in which GST was added instead ofGST-p53, followed by Western blot analysis. “GST-p53 Pull” representsthe results of the immunoprecipitation in which GST-p53 was added,followed by Western blot analysis.

FIG. 10 shows the results of detecting the interaction between striaminand p53 in vivo.

The vectors expressing different striamins conjugated to GFP wereintroduced into COS7 cells, and the recombinant proteins were expressedin the cells and reacted with endogenous p53. Cell extracts from thesecells were separated into two fractions, an NP-40 lysed fraction and anSDS lysed fractions. These fractions were immunoprecipitated byincubation with anti-p53 antibody, followed by addition of proteinA/G-Sepharose. The immunocomplex thus obtained was subjected toSDS-PAGE, followed by Western blotting using anti-GFP and anti-p53monoclonal antibodies (“p531C” in the figure).

Immunoprecipitation was carried out using control IgG instead ofanti-p53 antibody for the detection (“Control IgG IC” in the figure).Additionally, without carrying out the immunoprecipitation usinganti-p53 antibody, SDS-PAGE was performed on the cell lysate (10% of thetotal amount of the protein used for the immunoprecipitation), followedby detection using anti-GFP monoclonal antibody (“In put” in thefigure).

FIG. 11 shows the location of striamin on the mouse chromosome at themetaphase. Specific probes for striamin and chromosome 12 were madevisible as green fluorescence at the positions indicated by arrows.Striamin was localized on the 12C3 region.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be explained in detail below with referenceto examples, but is not to be construed as being limited thereto.

Example 1 Cell Culture

Normal mouse embryonic fibroblast cells (CMEF) derived from mouseCD1-ICR cell line, an immortalized clone (RS-4) established from CMEF,and NIH 3T3 cells, all of which were used for the comparison of theresearch on proteins and cloning, were cultured according to thedescription in the reference (Wadhwa et al. (1991), Mutat. Res.13.256:243-254). COS7 cells used for transient transformation werecultured in Dulbecco's modified Eagle's minimum essential mediumsupplemented with 10% fetal bovine serum.

Example 2 Cloning and Sequencing of cDNA

Comparing the Triton X-100 soluble cell membrane fraction from thenormal mouse cells (CMEF) with that from the immortalized cells (NIH3T3) revealed that an approximately 33 kDa protein is present in NIH 3T3cells but not in CMEF cells (Wadhwa et al. (1991), Mutat. Res.13.256:243-254). This protein was separated from the SDS polyacrylamidegel and used to generate the polyclonal antibody. The anti-p33 antibodygenerated was used to clone cDNA via immunoscreening of the cDNA libraryderived from RS-4 cells and constructed with lambda ZAPII vectors. Theresultant five clones obtained were characterized by partial sequencingusing the T3 and T7 primers for pBluescript vectors. While three of thefive clones were shown to be identical to the known genes, i.e., Fuschop(Rabbitts et al. (1993), Nat. Genet. 4:175-180; Kuroda et al. (1995),Am. J. Pathol. 147: 221-1227), G-utrophin (Blake et al. (1995), Proc.Natl. Acad. Sci. USA. 92:3697-3701) and dystrophin (Love et al. (1989),Nature 339:55-58), the remaining two clones did not have correspondingsequences in the nucleotide sequence database. The in vitro translatedproducts of these two novel clones, however, were not precipitated withanti-p33 antibody. This fact shows that these two clones are not relatedto the 33 kDa protein derived from the immortalized cells and initiallyidentified on SDS polyacrylamide gel. The inventors characterized clone#336, one of the clones isolated. The sequence of the cDNA clone wasdetermined by the dideoxy chain termination method, and the reaction wasanalyzed by an ABI 377 automated sequencer.

The whole sequence of the cDNA named #336, which was 2.4 kb in length,was obtained by a method using 3′-->5′ exonuclease III (TAKARA KiloSequencing Deletion Kit, TAKARA Shuzo) and primer walking. The 5′terminal of the clone was obtained by means of 5′ Marathon RACEpolymerase chain reaction (PCR) on the mouse skeletal muscle cDNA, usingthree sequences of primer-specific antisense genes: SP1 (SEQ ID NO:3:TGTCACTGCCACGCCTTCTCGGTGCGCAG), SP2 (SEQ ID NO:4:TCCCGGCTGCCCTTTGGCCCATCTTGTCCC), and SP3 (SEQ ID NO:5:TGAGAAAGCGTTAGACGCTCTCAGAGCCCT).

5′ Marathon RACE PCR was carried out according to the protocol in“Marathon-Ready™ cDNA Kit” (mouse skeletal muscle, catalogue #7456-1)(Clontech).

The complete sequence of the striamin cDNA thus obtained is shown inFIG. 1 and SEQ ID NO:1. No homologous sequence to this full-length cDNAwas identified through the DNA databank search. The cDNA encodes aprotein 149 amino acids in length (pI-10.2), and no counterpart havingsignificant homology to this protein was identified in the proteindatabase. No known motif that allows prediction of the function of theprotein was identified by cDNA analysis using BLAST, PROSITE, GCG, andPSORT programs. The 5′ untranslated region of striamin was found to havea “C/GAAAA” repeat, and the 3′ untranslated region was found to have a“GT” repeat. ProtPram analysis predicted that the protein hascharacteristics of soluble proteins with a standard hydrophobicity of0.5 and with aliphatic index of 0.74. Analysis by the ScanPrositeprogram revealed the presence of two protein kinase C phosphorylationsites, i.e., “SDR” (one letter codes of amino acids) at the position ofamino acid residues 45 to 47, and “SPK” (one letter codes of aminoacids) at the position of amino acid residues 78 to 80; a casein kinaseII phosphorylation site, i.e., “SGLD” (one letter codes of aminoacids/SEQ ID NO:6) at the position of amino acid residues 12 to 15; andtwo myristylation sites, i.e., “GNYYCC” (one letter codes of aminoacids/SEQ ID NO:7) at the position of amino acid residues 111 to 116 and“GTRWAK” (one letter codes of amino acids/SEQ ID NO:8) at the positionof amino acid residues 120 to 125. Other interesting characteristics ofthis protein would be its highly positive net charge, and the presenceof a large number of serine, leucine and proline residues, and fourcysteines. Based on the sequence analysis of the cDNA and protein,striamin was not characterized as a member of any known gene family.

Example 3 In Vitro Transcription and Translation

The predicted ORF was cloned into pBSSK and subjected to in vitrotranslation to verify the presence of the ORF within the given sequence.

Specifically, the ORF was amplified from RS-4 cells by reversetranscription-polymerase chain reaction, using a sense primer (SEQ IDNO:9: GAARRCATGAAAGGCCTGGCTGGCGAG) and an antisense primer (SEQ IDNO:10: GAATTCTCATGTCACTGCCACGCCTTCTCG), and the amplified product wascloned into Bluescript vectors.

In vitro transcription (Transprobe T kit, Pharmacia) and translation ofpBSSK/striamin were carried out for one hour in the rabbit reticulocytelysate (Stratagene) containing L-[³⁵S]methionine. The translatedproducts were separated on an SDS-polyacrylamide gel and visualized byautoradiography. In vitro translation products were alsoimmunoprecipitated with anti-p33 antibody. As a result, an approximately18 kDa protein was detected, and this agreed well with the proteinpredicted from the OFR within the sequence of #336 in the molecularweight (FIG. 1).

Example 4 Preparation of Recombinant Striamin Proteins

The open reading frame of striamin cDNA was amplified frompBSSK/striamin by PCR using a sense primer containing a Bam HI site (SEQID NO: 11: GGATCCAAGAAAGGCCTGGCTGGCGAG) and an antisense primercontaining a Hind III site (SEQ ID NO: 12: AAGCTTTCATGTCACTGCCACGCCTTC).The 0.5 kb fragment amplified by PCR was initially cloned into pGEM-Tvector, and the integrity of the sequence was confirmed. The sequencewas then excised with Bam HI and Hind III and finally cloned into pQE30vector that produces a His-tagged protein (Qiagen). The pQE30/striaminconstruct was introduced into M15 bacterium. After growing to OD580=0.6,the cells were induced by isopropyl-b-thiogalactopyranoside (IPTG) (0.2mM) for five hours at 37° C. The cell lysates of the bacterium (inducedand non-induced by IPTG) were analyzed by SDS-PAGE, and subsequently byWestern blotting using anti-His (Qiagen) and anti-p33 antibodies. Theresult revealed that an approximately 18 kDa protein was synthesized(FIG. 2). Similar to the result of the in vitro translation of the cDNAclone, the recombinant protein had the same size as was deduced from thesequence.

Example 5 Northern Blot Analysis

Northern blots, on which 2 μg/lane of poly (A+) RNA from a variety oftissues collected from humans and mice is blotted, were purchased from“Clontech Laboratories, Palo Alto, Calif.” Northern blot analysis wascarried out using 15 μg of total RNA prepared from each cell line. As aprobe, the 1.4 kb fragment of the 3′ untranslated region (UTR), whichwas recovered from the digestion with Bam HI of the plasmid #336, wasused. Hybridization was carried out in the SSC-Denhardt's-SDS buffer ata temperature of 65° C. After being washed twice in 2×SSC, 2×SSCcontaining 0.1% SDS, 1×SSC, and then 1×SSC containing 0.1% SDS for 10minutes each, the membrane was subjected to autoradiography to visualizethe result. The amounts of RNA loaded on the blot were determined byhybridization using β-actin or 18S ribosomal RNA as a probe. From theNorthern blot analysis of the expression of striamin in various tissuesfrom a number of mice and humans, intensive reactivity against a singletranscript of 3.0 kb from skeletal muscles of mice and humans wasdemonstrated (FIGS. 3A and B). A transcript of the same size was alsoshown to be expressed in the hearts of mice (FIG. 3A). Subsequently, theinventors examined whether this transcript is muscle fiber-typespecific. The four phenotypes of the fibers, i.e., fast-twitch fibers2A, 2B, and 2X, and type I slow-twitch fibers, are defined according toexpression of isoforms of myosin heavy chains (Pette and Staron (1990),Rev. Physiol. Biochem. Pharmacol. 116:1-76). Striamin expression wasmore marked in the fast muscle (quadriceps muscle of thigh) than in theslow muscle (soleus muscle) (FIG. 4A). Northern blot analysis of mouseskeletal muscles in which contents of fast and slow fibers are varied,including quadriceps muscle of thigh (95% fast-twitch fibers 2B, 4%fast-twitch fibers 2×; Hamalainen and Pette (1993), J. Histochem.Cytochem. 41:733-743), extensor digitorum longus muscle (60% fast-twitchfibers 2B, 28% fast-twitch fibers 2X, 12% fast-twitch fibers 2A;Leferovich et al. (1995), J. Neuroscience 15:596-603), outer layer ofthe gastrocnemius muscle (100% fast-twitch fibers 2B; Zardnnn and Parry(1994), Muscle & Nerve 17:1308-1316), diaphragm (57% fast-twitch fibers2X, 34% fast-twitch fibers 2A, 7% slow-twitch fibers; Zardnnn and Parry(1994), Muscle & Nerve 17:1308-1316), and soleus muscle (45% fast-twitchfibers 2A, 55% slow-twitch fibers; Lewis et al. (1982), J. Physiol.325:393-401), demonstrated that striamin is predominantly expressed inthe fiber 2B that has a fast glycolytic function (FIG. 4B).

The inventors then analyzed striamin expression during the myogenesis ofC2C12 myoblast cells in vitro. The striamin expression of the cellscultured in differentiation medium for four days was negligible,compared to the culture in non-differentiation medium and one dayculture in the differentiation medium (FIG. 4C).

Example 6 Expression Cloning and Biological Activity

The striamin ORF was amplified by PCR using a sense primer having a HindIII site (SEQ ID NO:13: GGTAAGCTTATATTGTTTGCAACTACCT), and an antisenseprimer having a Bam HI site (SEQ ID NO: 14:GGATCCCATGTGACCTAATGTTTCATGTCA). The fragment thus amplified wasinitially cloned into pGEM-T vector, and the integrity of the sequencewas confirmed. The insert was digested with Bam HI and Hind III, andthen incorporated into a mammalian expression vector LK444. This vectorhas a β-actin promoter for constitutive expression and a neo marker, andis essentially expressed in the mammalian cells cultured in the growthmedium containing G418 for selection (Gunning et al. (1987), Proc. Natl.Acad. Sci. U.S.A. 84:4831-4835). Mouse CIC12 myoblast cell line wastransformed using Lipofectamine (GIBCO-BRL), and the transformants wereselected in the medium containing G418 (700 μg/ml). Differentiation ofthe G418-resistant clones into myotubes in vitro was analyzed in amedium containing 2% equine serum.

As a result, the cells transformed with the vector were observed todifferentiate in the differentiation medium and develop myotubes.However, among eight clones transformed with selected #336, seven cloneswere not observed to develop myotubes to the same extent (FIG. 5). Thisresult revealed that overexpression of #336 inhibited thedifferentiation of C2C12 cells in vitro.

Example 7 Intracellular Localization of Striamin

The striamin ORF was inserted into the C-terminal of GFP ORF in pEGFPC1vector (Clontech) in frame. This plasmid, which encodes the GFP-striaminfusion protein, was introduced into COS7 cells proliferating on thecover glass by Lipofectamine™ (Gibco BRL). The cover glass was fixedwith a nuclear staining agent, Hoechst 33258 (Sigma) (5 to 10 μg/ml inthe culture medium for 10 minutes prior to the cell fixation), andmethanol/acetone (1:1). After washing with PBS three times, Fluoromount(Difco) was mounted on the cover glass. The cells were observed throughan epifluorescence optic system Olympus BH-2 microscope, or a ZeissAxiophot microscope coupled with a Cellscan System (Scanalytics, USA).As a result, the transformed cells exhibited distinct greenfluorescence, overlapping with the Hoechst 33258 nuclear staining in thesame cells (FIG. 6A).

Microinjections of pGFPC1/striamin, pGFPC1/N-terminal 75 amino acidresidues of striamin, and pGFPC1/C-terminal 74 amino acid residues ofstriamin were performed directly into the nuclei of NIH3T3 cells grownon the cover glass, using an Eppendorf microinjector and a Nikoninverted microscope. As described above, the cells were fixed and thenanalyzed for nuclear localization of striamin. As a result, distinctgreen fluorescence in the nucleus was detected for pEGFPC1-striamin,while it remains in the cytoplasm for both striamin-N75 and striamin-C74(FIG. 6B). As anticipated from amino acid composition of striamin, thedata and the fact that striamin does not contain any known nuclearlocalization signal suggest that the conformation of the intact proteincharged highly positively in the native form may be responsible fornuclear localization.

Example 8 Analysis of the Effect of Striamin on p53 Activity (LuciferaseAnalysis)

Wild-type p53 has been demonstrated to function during the celldifferentiation (Aloni-Grinstein et al. (1995), EMBO J. 14:1392-1401).The evidence to support this finding includes the facts that:

i) Over-expression of exogenous p53 or irradiation of cells canpartially recover the tumor cell differentiation (Halevy et al. (1995),Science 267:1018-1021),

ii) p53 mRNA is positively regulated during C2 differentiation, and

iii) inhibition of endogenous wild-type p53 suppresses the hematopoiesisand cell differentiation of muscles (Soddu et al. (1996), J. Cell Biol.134:193-204).

The role of p53 in the C2 differentiation is shown to be independent ofits activity exerted during the cell cycle (Soddu et al. (1996), J. CellBiol. 134:193-204). From this report's point of view, as well asconsidering the characteristics of striamin that localized in thenucleus and suppressed myogenic differentiation, the inventors suspectedthat striamin also has a certain effect on p53 activity. The inventorsthus examined whether striamin could inhibit p53 activity. For thisexamination the experiments were independently quadruplicated.

A temperature-sensitive p53 expression plasmid (pMSVp53Val135) andp53-responsive luciferase reporter (PG-13luc) plasmid, and one of thedifferent striamin expression vectors (a plasmid expressingLK444/full-length striamin-S, 75 amino acid residues at the N-terminal(LK444/N-striamin-S), or 74 amino acid residues at the C-terminal(LK444/C-striamin-S)), a plasmid expressing antisense RNA against thefull-length striamin cDNA (LK444/strimain-AS), or a control plasmid(LK444 vector (Gunning et al. (1987), Proc. Natl. Acad. Sci. U.S.A.84:4831-4835) were introduced into p53−/−mouse embryonic fibroblast(MEF) cells.

As shown in FIG. 7, the full-length striamin (sense-strand) reduced thep53 activity 4.6-fold, compared with the control. In contrast, antisensestriamin increased the p53 activity 1.6 fold, compared with the control.An inhibitory effect of C-striamin was equivalent to that of thefull-length striamin. However, the inhibitory effect on p53 activity wasnot detected for N-striamin. These results reveal that striamin canreduce the p53 transcriptional factor activity.

Example 9 Analysis of the Effect of Striamin on p53 Activity(β-Galactosidase Analysis)

p53−/−mouse embryonic fibroblast cells were microinjected with 0.1 μg/μlof each pMSVpS3Val135, and p53 responsive β-gal reporter pRGC fos-lacZ(Oncogene, 1998, 16: 3317-3322), and LK444 (control), LK444/full-lengthstriamin, or LK444/striamin-AS. To identify the cells successfullyinjected with DNA, a control IgG was also microinjected into the cellsin conjunction with the plasmids above. After overnight culture at 32.5°C., the cells were fixed with 4% formaldehyde and treated with PBScontaining 0.1% Triton X-100 on ice for five minutes to make the cellspermeable to macromolecules, followed by washing three times with PBS.

Subsequently, the cells were stained with FITC-conjugated anti-rabbitIgG (the upper panels) in order to specify the cells into which the DNAwas introduced. The stained cells are indicated by the arrows in thelower panels.

Detection of the effect of striamin on p53 activity (β-galactosidaseexpression) was conducted using a β-galactosidase staining kit(Boehringer Mannheim). The cells were observed by microscopy (Proris(AX70), Olympus). Blue cells were determined as positive for β-galexpression.

As a result, the cells into which a control plasmid (LK444) and anantisense striamin plasmid (LK444/striamin-AS) were introduced yielded86% and 88% β-gal positive cells, respectively.

In contrast, only 5% of the cells into which the striamin sense plasmid(LK444/full-length striamin) was introduced were detected as positive(FIG. 8). Consequently, this study also supports that striamin inhibitsp53 activity. This is consistent with the fact that striamin expressionis accompanied by a reduction of C2C12 cell differentiation.

Example 10 Interaction Between Striamin and p53 In Vitro

That striamin has an effect on the function of p53 to activatetranscriptions suggests the possibility of interaction between the twoproteins. To study if striamin interacts with p53 in vitro,immunoprecipitation, followed by Western blotting, was carried out.

(1) Preparation of Different Striamins with a Histidine Tag Attached(Fusion Proteins)

The DNAs encoding the full-length striamin, and N-terminal (75 aminoacid residues) and C-terminal (74 amino acid residues) regions ofstriamin were inserted into an expression vector pQE30 (Qiagen) of E.coli to generate pQE30/full-length striamin, pQE30/N-striamin, andpQE30/C-striamin. These plasmids were introduced into E. coli, and therecombinant proteins were expressed and then purified. Specifically, thecells were first centrifuged, and their pellet was suspended in Buffer A(10 mM Tris-Cl (pH 7.5), 150 mM NaCl, 20 mM imidazole, 6 M urea, and 5mM β-mercaptoethanol) and was sonicated on ice for two minutes, followedby agitation for 30 minutes at room temperature. The extract wassubjected to centrifugation at 15,000 g for 20 minutes. The supernatantwas applied to the nickel-NTA-agarose affinity resin (0.5 ml) (Qiagen)and mixed for two hours at room temperature. The mixture was chargedinto the column, which was subsequently washed with 20 ml of TBS (10 mMTris-Cl (pH 7.5) and 0.5 M NaCl (TBS)). The proteins adsorbed into theaffinity resin were eluted with TBS solution containing 0.5 M imidazole,and the imidazole was removed using a PD-10 column (Pharmacia). Thedifferent striamins thus obtained were stored at −20° C. until use. Thepurity of the purified products was identified by SDS-PAGE and Westernblot analysis using anti-His antibody.

(2) Analysis of Binding Between Different Striamins and p53

Each protein purified in step (1) (2 to 5 μg) was mixed with GST orGST-p53 (1 μg, Santa Cruz) in NP40-lysis buffer (500 μl). After twohours, glutathione-Sepharose beads (20 μl) were added to the suspensionand rotated for mixing for one hour at 4° C. The beads were precipitatedby centrifugation, washed three times with TBS and then boiled inSDS-sample buffer. Subsequently, SDS-PAGE was carried out for eachsample, followed by detection of the respective striamins conjugated tothe histidines by Western blotting using an anti-histidine-tag antibody.In addition, p53 was detected by Western blotting using anti-p53antibody.

As a result, the respective striamins conjugated to histidines wereshown to precipitate in the GST-p53 precipitation usingglutathione-Sepharose beads (FIG. 9, Lanes 7 to 9). In conclusion, thefull-length striamin, N-striamin, and C-striamin all interacted withp53.

In addition, to confirm the striamin expression in all the samples,samples without addition of GST were subjected to Western blot analysisas controls (Lanes 1 to 3). Striamin was shown to be expressed in eachsample.

To verify that the striamins are not directly bound to GST, similardetection was conducted adding GST instead of GST-p53 (Lanes 4 to 6).The respective striamins were not bound to GST directly.

Example 11 Interaction Between Striamin and P53 In Vivo

COS7 cells with high transfection efficiency (expressing endogenous p53)were transfected with expression plasmids for the fusion proteins of GFPand either of the striamins (pEGFPC1/full-length striamin,pEGFPC1/N-striamin, or pEGFPC1/C-striamin), or the control (pEGFPC1vector). The cell extracts were prepared after 48 hours. A solubleprotein fraction in NP-40 cell lysis buffer was obtained in thepreparation of the cell extracts. From the insoluble fraction in theNP-40 cell lysis buffer, a soluble fraction was obtained after additionof 0.5% SDS to this fraction and boiling it.

To each of these fractions, anti-p53 antibody (CM-1, NovocastraLaboratories Ltd.) was added. After incubating overnight at 4° C.,protein A/G-Sepharose was added to the mixtures and allowed to react for30 minutes at 4° C., followed by immunoprecipitation (centrifugation;5000 rpm for 1 min.).

The immunocomplexes thus obtained were subjected to SDS-PAGE and thenWestern blotting using an anti-GFP monoclonal antibody (#8362-1,Clontech). Western blotting using anti-p53 monoclonal antibody (Ab-1,Calbiochem) was also carried out to detect the presence of p53.

The result revealed that the precipitation of endogenous p53 usinganti-p53 polyclonal antibody (CH-1, Novocastra Laboratories Ltd.)coprecipitated the respective striamins bound to GFP (the right sidelanes in FIG. 10). In particular, striamins were detected in the SDSsoluble fractions (the right side lanes in FIG. 10, Lanes 6 to 8). Thisshowed that striamin interacts with p53 intracellularly in COS cells.The endogenous p53 failed to bind to GFP (the right side lanes in FIG.10, Lane 5).

In contrast, the striamins were not detected with immunoprecipitationusing control IgG, instead of anti-p53 antibody, followed by Westernblotting using anti-GFP monoclonal antibody (the central lanes in FIG.10). However, GFP and GFP-bound striamin bands were detected as a resultof SDS-PAGE of the cell lysate (10% volume of the protein used forimmunoprecipitation), without undergoing immunoprecipitation usinganti-p53 antibody, followed by Western blotting using anti-GFPmonoclonal antibody (the left side lanes in FIG. 10).

Example 12 Location on the Chromosome

Mouse P1 genomic clones were obtained via PCR screening of the P1bacteriophage mouse genomic library, using #336 specific primers, i.e.,a sense primer (SEQ ID NO:15: TGGTATTCTTATATTGTTTGCAACTAACTA) and anantisense primer (SEQ ID NO:16: GGAAGGCCATGTGACCTAATGTTTCATGTCA). P1clones isolated were tested for hybridization with the 3′ UTR region ofthe gene, subsequently used for determining their location on thechromosome by fluorescence in situ hybridization (FISH). DNA from amouse P1 clone was labeled with digoxigenin-dUTP by nick-translation.After binding to mouse DNA cleaved in pieces, the labeled probe washybridized to the metaphase chromosome derived from mouse embryonicfibroblast cells in solution containing 50% formamide, 10% dextransulfate, and 2×SSC. After hybridization, the slide glass was incubatedwith fluorescence labeled anti-digoxigenin antibody, and the specifichybridization signal was detected by counter-staining with4′,6′-diamidino-2-phenylindole (DAPI). As a result, the medium-sizedchromosome, which was considered to be chromosome 12 based on DAPIstaining, was specifically labeled. In the second experiment, a specificprobe for a centromere region of chromosome 12 was hybridized with theP1 clone. Striamin P1 was located on chromosome 12 (FIG. 8). A total of80 metaphase cells were analyzed and 71 of these cells were specificallylabeled. In particular, the measurements of the 10 cells that hybridizedwith chromosome 12 have revealed that striamin located on the 57% distalposition from the boundary between heterochromatin and euchromatintoward the telomere region of chromosome 12, namely, toward the regionassociated with the band 12C3 (corresponding to human chromosome14q21-22).

INDUSTRIAL APPLICABILITY

The present invention provides striamin protein that inhibitsdifferentiation of myoblasts into myotubes, and the gene encodingstriamin. As the striamin protein and gene of the present invention arethought to play a role in maintaining the cultured cells in anundifferentiated state, they are expected to be applied to, for example,cancer therapy. For the mouse and human genes, intense expression isfound in heart and skeletal muscle at the tissue level, and applicationsto diseases associated with heart or muscle are contemplated. Moreover,the mouse and human genes are specifically expressed in the fast-twitchfibers of muscle fibers. Since the mechanism involved in the phenotypesof slow and fast muscle fibers still remains an unsolved question,analysis of the gene of the present invention is expected to showapplicability to movement.

1-33. (canceled)
 34. An isolated antibody that specifically binds to apolypeptide the amino acid sequence of which is set forth in SEQ IDNO:1.
 35. The isolated antibody of claim 34, which is a monoclonalantibody.
 36. The isolated antibody of claim 34, which is a polyclonalantibody.
 37. The isolated antibody of claim 34, which is a chimericantibody.
 38. The isolated antibody of claim 34, which is a humanizedantibody.
 39. An isolated antibody that specifically binds to apolypeptide the amino acid sequence of which consists of residues 76through 149 of SEQ ID NO:1.
 40. An isolated antibody that specificallybinds to a polypeptide the amino acid sequence of which consists ofresidues 1 through 75 of SEQ ID NO:1.
 41. An isolated Fab fragment thatspecifically binds to a polypeptide the amino acid sequence of which isset forth in SEQ ID NO:1.
 42. An isolated antibody, or portion thereof,produced by immunizing an animal with a polypeptide comprising at least30 contiguous amino acid residues of SEQ ID NO:1, wherein said antibodyor portion thereof specifically binds to the amino acid sequence of SEQID NO:1.
 43. The isolated antibody, or portion thereof, of claim 42,wherein the polypeptide comprises at least 50 contiguous amino acidresidues of SEQ ID NO:1.
 44. The isolated antibody, or portion thereof,of claim 42, wherein the polypeptide comprises the amino acid sequenceof residues 1 through 7 of SEQ ID NO:1.
 45. The isolated antibody, orportion thereof, of claim 42, wherein the polypeptide comprises theamino acid sequence of residues 76 through 149 of SEQ ID NO:1.
 46. Theisolated antibody, or portion thereof, of claim 42, wherein thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:1.47. The isolated antibody, or portion thereof, of claim 42, wherein theanimal is a rabbit.
 48. The isolated antibody, or portion thereof, ofclaim 42, wherein the animal is a mouse.
 49. The isolated antibody, orportion thereof, of claim 42, wherein the antibody is polyclonal. 50.The isolated antibody, or portion thereof, of claim 42, wherein theantibody is monoclonal.
 51. A hybridoma cell line that produces amonoclonal antibody that binds to a polypeptide the amino acid sequenceof which is set forth in SEQ ID NO:1.
 52. A method for detecting astriamin polypeptide in a sample, the method comprising: a) contactingthe sample with the antibody of claim 34; and b) detecting binding ofthe antibody to a component of the sample, wherein said binding isindicative of the presence of striamin polypeptide in the sample. 53.The method of claim 52, wherein the antibody is detectably labeled.